RLS dissection
this page is a work in progress* *Patching (Note: do not perform without being specifically trained for patching ) **Always make sure to use sterile toothpicks (these should have autoclave tape somewhere on them, usually the lid, and black lines in the autoclave tape indicating sterility). Be sure you are not using used toothpicks. **Match the strain # from the lifespan printout sheet to each ID # for the RLS. Typically you will patch 3 strains per plate. If there are any significant questions or confusion, call the person who submitted the lifespan and get clarification (see the contact sheet for phone numbers). *Setup ** *Incubation times **New lifespans will be incubated for 2 hours. After the majority of cells are no longer dividing enough at 2hrs, then the time can be increased to 3 hours. Similarly, eventually the incubation time can be increased to 4 hours. Let all lifespans get at least 4 hours at 30C each day (i.e., count 2hr lifespans at least twice). *Dissecting cells **You will acquire hands on training on how to dissect yeast cells (typically at least 10 hours of training is required). *Entering data **For any duration incubated lifespan, only 1 cell (just the mother present) = 0 **For any duration incubated lifespan, 2 total cells (mother plus one daughter), = 1 division **For a lifespan incubated 2 hr, 2-5 = 2 divisions; ≥'6' cells = 3 divisions of the mother; ≥15 = 4 **For a lifespan incubated 3 hr, ≥'8' cells = 3 divisions of the mother; ≥20 = 4 **For a lifespan incubated 4 hr, ≥'10' cells = 3 divisions of the mother; ≥25 = 4 *Potential problems **Lost cells ***Sometimes there will be a "ghost" cell, and it may just be hard to see but not lost. If a cell is missing, first determine exactly which cell is missing. Sometimes there will be a gap where the cell used to be, or sometimes you can see the spot on the plate where the needle has created an indentation where the cell once was. On the excel sheet, write "LOST" (without quotation marks) instead of the cell division number for that round. Punch a hole in the place where the cell was to indicate it was lost. From then on, when encountering this spot on the excel sheet when counting, simply skip over it. **colonies or contamination on plate ***Make note of the contamination at the top of the spreadsheet in the lifespan plate "notes" section, be reasonably detailed. ***If the contamination is on top of some mother cells. Count those cells as LOST and for mothers near contamination, move the remaining mothers to another location far enough from the contamination so that the contamination will again grow into the mothers. Make an arrow and maybe a line with holes to help guide the next dissectors to where the mothers were moved. ***If the contamination is far enough from mothers: (use your judgement, and remember that contamination can spread). Depending on the position, p'''ossibly '''remove the contamination using sterile technique (only do this if you have been trained, or ideally you will give the plate to Daniel or someone experienced to remove the contamination) *Killing strains *Killing plates *Killing lifespans